1. Volume 15, Issue 4, Pages 505-517 (April 2012)
Yin Yang 1 Deficiency in Skeletal Muscle Protects against Rapamycin-Induced Diabetic- like Symptoms through Activation of Insulin/IGF Signaling 
Sharon M. Blättler, John T. Cunningham, Francisco Verdeguer, Helen Chim, Wilhelm Haas, Huifei Liu, Klaas Romanino, Markus A. Rüegg, Steven P. Gygi, Yang Shi, Pere Puigserver 
Cell Metabolism 
Volume 15, Issue 4, Pages (April 2012)
DOI: /j.cmet
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2. Figure 1
Chronic Rapamycin Treatment Causes Insulin Resistance and Lipid Dysregulation
(A–G) Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. Shown are: glucose tolerance test (A), insulin tolerance test (B), serum insulin level and insulin/C-peptide ratio (C), glucose infusion rate during hyperinsulinemic-euglycemic clamps (D), glucose uptake rate under basal and clamp condition and index of tissue glucose uptake (Rg) (E), serum triglycerides and cholesterol (F), and intramyocellular triglycerides (G). All values are presented as mean ± SD, n = 6–10, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figures S1 and S2.
Cell Metabolism  , DOI: ( /j.cmet )
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3. Figure 2
Chronic Rapamycin Treatment Decreases Insulin Signaling in Skeletal Muscle and Liver
(A–C) Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days, fasted for 12 hr, and then injected with vehicle or 0.6 U/kg insulin 10 min before sacrifice. Insulin signaling in skeletal muscle (A) and liver (B) are shown. Also shown is gene expression in the soleus from fed mice (C). All values are presented as mean ± SD, n = 6–10, ∗p < 0.05, and ∗∗p < 0.01.
Cell Metabolism  , DOI: ( /j.cmet )
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4. Figure 3
Skeletal Muscle-Specific YY1 Knockout Mice Are Protected from Rapamycin-Induced Diabetic-like Effects
(A and B) Glucose tolerance test (A) and insulin tolerance test (B) in YY1mKO mice or wild-type littermates.
(C–G) Wild-type or YY1mKO mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. Shown are: glucose tolerance test (C), insulin tolerance test (D), serum cholesterol and triglycerides (E), serum insulin levels (F), and intramyocellular triglycerides (G). All values are presented as mean ± SD, n = 6–10, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S3.
Cell Metabolism  , DOI: ( /j.cmet )
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5. Figure 4
Skeletal Muscle-Specific YY1 Knockout Mice Display Hyperactivation of Insulin/IGF Signaling and Increased Gene Expression that Are Insensitive to Rapamycin Treatment
(A and B) Wild-type or YY1mKO mice were treated for 14 days with vehicle or 2.5 mg/kg rapamycin, fasted for 12 hr, and then injected with vehicle or 0.6 U/kg insulin 10 min before sacrifice. Insulin signaling in skeletal muscle is shown.
(C) Wild-type or YY1mKO mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days and gene expression in the soleus of fed mice was measured.
(D) Gene expression in C2C12 myotubes infected with scrambled shRNA or YY1 shRNA for 72 hr.
(E) Gene expression in C2C12 myotubes infected with GFP or Flag-YY1 for 48 hr. All values are presented as mean ± SD, n = 6–10, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <  See also Figure S4.
Cell Metabolism  , DOI: ( /j.cmet )
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6. Figure 5
Rapamycin Suppresses Insulin/IGF Signaling Genes by Promoting Interaction between YY1 and the Polycomb Corepressor Pc2
(A) Coimmunoprecipitation and western blot analysis in HEK293 cells.
(B) YY1 protein cartoon.
(C) Coimmunoprecipitation and western blot analysis in HEK293 cells.
(D) Luciferase assay in HEK293 cells with a Gal4-YY1 luciferase construct and the indicated polycomb proteins.
(E) Coimmunoprecipitation of HA-YY1 and Flag-Pc2 in HEK293 cells treated with vehicle or rapamycin (20 nM) for 2 hr.
(F) Endogenous interaction between YY1 and Pc2 was detected in skeletal muscle from mice treated with vehicle or 2.5 mg/kg rapamycin for 2 hr.
(G) Gene expression in C2C12 myotubes infected with GFP or Flag-Pc2 for 48 hr.
(H) Coimmunoprecipitation and western blot analysis of HEK293 cells treated with vehicle or rapamycin (20 nM) for 2 hr.
(I) Gene expression in C2C12 myotubes infected with GFP, wild-type YY1, YY1-AA, or YY1–DD for 48 hr. All values are presented as mean ± SD, n = 6, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figures S5 and S6.
Cell Metabolism  , DOI: ( /j.cmet )
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7. Figure 6
Rapamycin Induces Recruitment of YY1 and Pc2 to Promoters of Insulin/IGF Signaling Genes that Are Associated with H3K27 Trimethylation
(A–D) Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. ChIP was performed in whole tissue extracts from skeletal muscle of refed mice using specific antibodies for YY1 (A), Pc2 (B), and H3K27 trimethylation (C and D). All values are presented as mean ± SEM, n = 4, ∗p < IGF2 neg is a negative control in the Igf2 gene promoter, which does not contain YY1 binding sites.
Cell Metabolism  , DOI: ( /j.cmet )
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8. Figure 7
YY1 Suppresses Insulin/IGF Signaling Genes through the Polycomb Proteins Pc2 and Ezh2
(A) Mice were treated with vehicle or 2.5 mg/kg rapamycin for 14 days. ChIP was performed in whole tissue extracts from skeletal muscle of refed mice using specific antibodies for Ezh2. All values are presented as mean ± SEM, n = 4.
(B) Coimmunoprecipitation and western blot analysis of HEK293 cells treated with vehicle or rapamycin (20 nM) for 2 hr.
(C) shScrambled-, shPc2-, or shEzh2-stable C2C12 myotubes were infected with GFP, wild-type YY1, YY1-AA, or YY1-DD for 48 hr, and gene expression was measured. All values are presented as mean ± SD, n = 6, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
(D) Active mTORC1 induces YY1 phosphorylation at T30 and S365, resulting in displacement of Pc2, thereby activating insulin/IGF signaling gene transcription. Conversely, inactive mTORC1, such as in the presence of rapamycin, results in YY1 dephosphorylation at T30 and S365, permitting recruitment of Pc2 and consequently the polycomb repressor complex (PRC) to inhibit expression of insulin/IGF signaling genes. The recruitment of YY1 and Pc2 at these promoters correlates with an increased level of H3K27 trimethylation produced by Ezh2, which is a marker of transcriptional repression. In the absence of YY1, the suppression on these genes is relieved, leading to their hyperactivation and rapamycin insensitivity. Ac, acetylation; Me, methylation; TAC, transcriptional activator complex.
Cell Metabolism  , DOI: ( /j.cmet )
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